Method of extracting volatile compounds from tobacco material

ABSTRACT

There is provided a method of extracting one or more volatile compounds of interest from tobacco material, the method comprising the steps of: i) providing tobacco material; ii) subjecting the tobacco material to steam distillation; and iii) extracting one or more volatile compounds of interest from the tobacco material with a solvent; wherein distillation step (ii) and extraction step (iii) are carried out simultaneously and at a pH of no greater than 2, and wherein the period during which both the distillation step (ii) and the extraction step (iii) are carried out is from about 8 to about 20 hours.

FIELD

The present invention relates to a method of extracting one or morevolatile compounds of interest from tobacco material, a tobacco extractobtainable or obtained by said method, a tobacco product comprising saidtobacco extract, a method of preparing said tobacco product, and the useof said tobacco extract.

BACKGROUND

Popular smoking articles, such as cigarettes, have a substantiallycylindrical rod shaped structure and include a column of a smokablematerial such as tobacco (e.g., in cut filler form) surrounded by apaper wrapper thereby forming a so-called “tobacco rod”. Normally, acigarette has a cylindrical filter element aligned in an end-to-endrelationship with the tobacco rod and the filter element is attached toone end of the tobacco rod using a circumscribing wrapping materialknown as tipping paper. A cigarette is employed by a smoker by lightingone end of the cigarette and burning the tobacco rod. The smoker thenreceives a mainstream smoke into his/her mouth by drawing on theopposite end (e.g., the filter end) of the cigarette.

New smoking articles are also being designed and launched in the marketas new generation products. These can be classified into three maingroups and may be referred to as “vaping articles”:

-   -   1. Electronic devices;    -   2. Heating devices; and,    -   3. Hybrid devices.

Hybrid devices are a combination of an electronic and heating device.

Through the years, various treatment methods and additives have beenproposed for altering the overall character or nature of tobaccomaterials utilized in tobacco products. For example, additives ortreatment processes have been utilized to alter the chemistry or sensoryproperties of the tobacco material in cigarettes or in the case ofsmoking or vaping tobacco materials, to alter the chemistry or sensoryproperties of mainstream smoke/aerosol generated by smoking/vapingarticles including the tobacco material.

The sensory attributes can for instance be enhanced by incorporatingflavouring materials into various components of the cigarette orsmoking/vaping article. Exemplary flavouring additives include mentholand products of Maillard reactions, such as pyrazines, amino sugars, andAmadori compounds.

There is, however, still a need in the art for compositions suitable foraddition to tobacco products (which may include smoking and/or vapingproducts) to introduce desired sensory characteristics. In particular,it would be desirable to provide a method for efficient extraction andisolation of such compositions.

SUMMARY

In accordance with some embodiments described herein, a method ofextracting one or more volatile compounds of interest from tobaccomaterial is provided, the method comprising the steps of:

-   -   i) providing tobacco material;    -   ii) subjecting the tobacco material to steam distillation; and    -   iii) extracting one or more volatile compounds of interest from        the tobacco material with a solvent;        wherein distillation step (ii) and extraction step (iii) are        carried out simultaneously and at a pH of no greater than 2, and        wherein the period during which both the distillation step (ii)        and the extraction step (iii) are carried out is from about 8 to        about 20 hours.

In accordance with some embodiments described herein, a tobacco extractobtainable or obtained by a method as defined herein is provided,wherein the tobacco extract comprises one or more volatile compounds ofinterest in an amount of at least about 75% by weight of the tobaccoextract.

In accordance with some embodiments described herein, a tobacco productis provided, the tobacco product comprising a tobacco extract as definedherein.

In accordance with some embodiments described herein, a method ofpreparing the tobacco product as defined herein is provided, the methodcomprising the steps of:

-   -   (a) preparing a tobacco extract in accordance with a method as        defined herein; and    -   (b) combining the tobacco extract directly with a tobacco        product and/or combining the tobacco extract with reconstituted        tobacco and optionally combining the reconstituted tobacco with        a tobacco product.

In accordance with some embodiments described herein, the use of thetobacco extract defined herein for improving the sensory properties of atobacco product is provided.

BRIEF DESCRIPTION OF THE FIGURES

Embodiments of the present invention are described, by way of exampleonly, with reference to the accompanying drawings in which:

FIG. 1 shows a Likens-Nickerson apparatus for simultaneousdistillation-extraction.

FIG. 2 shows a simultaneous distillation-extraction apparatus, which isuseful when the solvent is more dense than water.

FIG. 3 shows a simultaneous distillation-extraction apparatus used inExample 1, which is useful when the solvent is less dense than water.

FIG. 4 shows the simultaneous distillation-extraction apparatus used inExample 2, which is useful when the solvent is less dense than water.

FIG. 5 shows the simultaneous distillation-extraction apparatus of FIG.3 that has been enlarged in order to extract compounds from a 2240 gsample of tobacco.

FIG. 6 is a reference Figure that shows an example chromatogram in whichthe peaks are separated into three groups: volatile flavours,semi-volatile flavours, and diterpenes. This Figure is included for easeof interpretation only.

FIG. 7 shows the chromatograms obtained using the simultaneousdistillation-extraction method at (a) pH 2.0 (in accordance with theinvention) and (b) pH 6.0 (outside the scope of the invention).

FIG. 8 shows the chromatogram obtained using the simultaneousdistillation-extraction method at pH 6.0 (outside the scope of theinvention).

FIG. 9 shows the chromatogram obtained using the simultaneousdistillation-extraction method at pH 4.0 (outside the scope of theinvention).

FIG. 10 shows the chromatogram obtained using the simultaneousdistillation-extraction method at pH 3.0 (outside the scope of theinvention).

FIG. 11 shows the chromatogram obtained using the simultaneousdistillation-extraction method at pH 2.0 (in accordance with theinvention).

FIG. 12 shows the chromatogram obtained using the simultaneousdistillation-extraction method at pH 0.5 (in accordance with theinvention).

DETAILED DESCRIPTION

Method

A first aspect of the invention provides a method of extracting one ormore volatile compounds of interest from tobacco material, the methodcomprising the steps of:

-   -   i) providing tobacco material;    -   ii) subjecting the tobacco material to steam distillation; and    -   iii) extracting one or more volatile compounds of interest from        the tobacco material with a solvent;        wherein distillation step (ii) and extraction step (iii) are        carried out simultaneously and at a pH of no greater than 2, and        wherein the period during which both the distillation step (ii)        and the extraction step (iii) are carried out is from about 8 to        about 20 hours.

As used herein, the term “tobacco material” refers to a material derivedfrom a plant of the Nicotiana species. The selection of the plant of theNicotiana species is not limited, and the types of tobacco or tobaccosused may vary.

In some embodiments, the tobacco material is selected from flue-cured orVirginia, Burley, sun-cured, Maryland, dark, dark-fired, dark air cured,light air cured, Indian air cured, Red Russian and Rustica tobaccos, andmixtures thereof, as well as various other rare or specialty tobaccos,green or cured. Tobacco material produced via any other type of tobaccotreatment which could modify the tobacco taste, such as fermentedtobacco or genetic modification or crossbreeding techniques, is alsowithin the scope of the present invention. For example, it is envisagedthat tobacco plants may be genetically engineered or crossbred toincrease or decrease production of components, characteristics orattributes.

In some embodiments, the tobacco material is sun-cured tobacco, selectedfrom Indian Kurnool and Oriental tobaccos, including Katerini, Prelip,Komotini, Xanthi and Yambol tobaccos. In some embodiments, the tobaccomaterial is dark air cured tobacco, selected from Passanda, Cubano,Jatin and Bezuki tobaccos. In some embodiments, the tobacco material islight air cured tobacco, selected from North Wisconsin and Galpaotobaccos.

In some embodiments, the tobacco material is a cured or non-curedmixture of flue-cured, Burley and Oriental tobaccos.

For the preparation of smoking/vaping or smokeless tobacco products,plants of the Nicotiana species may be subjected to a curing process.Certain types of tobaccos may be subjected to alternative types ofcuring processes, such as fire curing or sun curing. Preferably, but notnecessarily, harvested tobaccos that are cured are aged.

The tobacco can be harvested in different stages of growth, for examplewhen the plant is about to sprout, produce leaves or even when it startsflowering.

In some embodiments, at least one portion of the plant of the Nicotianaspecies (e.g., at least a portion of the tobacco material) is employedin an immature form. That is, in some embodiments, the plant, or atleast one portion of that plant, is harvested before reaching a stagenormally regarded as ripe or mature.

In some embodiments, at least a portion of the plant of the Nicotianaspecies (e.g., at least a portion of the tobacco material) is employedin a mature form. That is, in some embodiments, the plant, or at leastone portion of that plant, is harvested when that plant (or plantportion) reaches a point that is traditionally viewed as being ripe,over-ripe or mature, which can be accomplished through the use oftobacco harvesting techniques conventionally employed by farmers. BothOriental tobacco and Burley tobacco plants can be harvested. Also, theVirginia tobacco leaves can be harvested or primed by stalk position.

The Nicotiana species may be selected for the content of variouscompounds that are present in the plant. For example, plants may beselected on the basis that those plants produce relatively highquantities of one or more of the compounds desired to be isolated (i.e.the volatile compounds of interest). In certain embodiments, plants ofthe Nicotiana species are specifically cultivated for their abundance ofleaf surface compounds. Tobacco plants may be grown in green-houses,growth chambers, or outdoors in fields, or grown hydroponically.

Various parts or portions of the plant of the Nicotiana species may beemployed in the method defined herein. In some embodiments, the wholeplant, or substantially the whole plant, is harvested and employed assuch. As used herein, the term “substantially the whole plant” meansthat at least 90% of the plant is harvested, such as at least 95% of theplant, such as at least 99% of the plant. Alternatively, in someembodiments, various parts or pieces of the plant are harvested orseparated for further use after harvest. In some embodiments, thetobacco material is selected from the leaves, stems, stalks of theplant, and various combinations of these parts. The tobacco material ofthe invention may thus comprise an entire plant or any portion of aplant of the Nicotiana species.

In some embodiments, the tobacco material comprises tobacco leaf. Insome embodiments, the tobacco material comprises tobacco leaf that iswhole or cut tobacco leaf. In some embodiments, the tobacco materialcomprises whole tobacco leaf. In some embodiments, the tobacco leafcomprises cut tobacco leaf. In some embodiments, the tobacco material ismilled tobacco.

In some embodiments, the tobacco material is stored at temperaturesbelow 0° C. before distillation and extraction. Therefore, in someembodiments, the tobacco material is kept frozen before distillationstep (ii) and extraction step (iii) are carried out. In someembodiments, the tobacco material is kept frozen, and is then defrosteduntil it reaches approximately room temperature (around 22° C.) beforedistillation step (ii) and extraction step (iii) are carried out.

In some preferred embodiments, the tobacco material does not undergo anyheat treatment before distillation step (ii) and extraction step (iii)are carried out. For example, in some embodiments, the tobacco materialis not heated to a temperature of greater than about room temperaturebefore distillation step (ii) and extraction step (iii) are carried out.It has been found that, when the tobacco material is not subjected toany heat treatment prior to distillation step (ii) and extraction step(iii) being carried out, the loss of volatile compounds important forthe extract flavour and taste prior to the carrying out of said steps(ii) and (iii) may be reduced.

The carrying out of the distillation step (ii) and extraction step (iii)simultaneously is a process known generally in the art as simultaneousdistillation-extraction (SDE). SDE provides a technique in which thesteps of isolation and extraction of certain compounds from a sample canbe achieved simultaneously.

It is generally accepted that the SDE method was created in 1964 byLikens and Nickerson who designed an original device for the analysis ofhop oil (Likens S T, Nickerson G B, ASBC Proc., 1964, 5; A. Chaintreau,Flavour and Fragrance Journal, 2001, 16: 136-148). In this method, theprocesses of steam distillation and extraction of volatiles from asample into a small quantity of solvent are combined. In the originalLikens-Nickerson method, a device 1 as shown in FIG. 1 was used toanalyse hop oil. The sample (an aqueous solution or a slurry of a solidmaterial in water) was boiled under stirring in the flask connected tothe left arm 2. Volatiles were then steam-distilled through the upperpart of the left arm 2 and, simultaneously, solvent vapours distilledthrough the upper part of the right arm 3. Vapours condensed on the coldfinger 4, and the extraction process began between both liquid films onthe condenser surface 5.

SDE has been found to have utility in the isolation of volatilesubstances from natural products. In this technique, the material fromwhich the volatile compounds should be removed is subjected to steamdistillation and, simultaneously, the compounds distilled are extractedwith a solvent.

A typical apparatus used for SDE is shown in FIG. 2 , noting that thisapparatus shown is useful for the SDE method where the solvent used ismore dense than water. As shown in FIG. 2 , the apparatus 10 includes aflask 11 for the tobacco sample, a flask 12 for the solvent, water baths13 and a heating/stirring plate 14 for heating the samples. Theapparatus 10 may typically also include an auxiliary heating coil 15 forheating the tobacco sample to a higher temperature than the solvent suchthat steam distillation may be carried out. Each of tubes 16 a, 16 bhave an internal diameter of about 4 mm. During the process, vapourscondense on the cold finger 17, and the extraction process may beginbetween both liquid films on the condenser surface 18. The apparatusused allows the return of the water and solvent distilled to theirrespective flasks, after the partitioning process. This makes itpossible to obtain, by the end of the process, an extract that is readyto be analysed by techniques such as gas chromatography.

In some embodiments, the distillation step (ii) and extraction step(iii) (i.e. SDE) are carried out at a pH of no greater than 2 for thetotal period of time for which said steps are carried out. It wassurprisingly found by the present inventors that, when a pH of nogreater than 2 is used during the SDE process, the amount of volatilecompounds extracted from the tobacco material is increased.

In some embodiments, the solvent is a non-polar solvent. In someembodiments, the solvent is a polar solvent. As used herein, the term“polar solvent” refers to any solvent having a dielectric constant ofgreater than or equal to 15. As used herein, the term “non-polarsolvent” refers to any solvent having a dielectric constant of less than15.

In some embodiments, the solvent is immiscible with water.

In some embodiments, the solvent comprises an organic-based solvent. Insome embodiments, the solvent is an organic solvent. In someembodiments, the solvent is a non-polar organic solvent. The term“organic” is understood by those skilled in the art. Typically, anorganic solvent is considered to be a solvent comprising carbon.

In some embodiments, the solvent is a non-polar solvent that isimmiscible with water.

In some embodiments, the solvent is selected from the group consistingof n-butanol, cyclohexane, dichloromethane, ethyl acetate, heptane,hexane, methyl-t-butyl ether, 2-butanone, pentane, diisopropyl ether,diethyl ether, and mixtures thereof. In some embodiments, the solvent isselected from the group consisting of pentane, diethyl ether andmixtures thereof. In some preferred embodiments, the solvent is amixture of pentane and diethyl ether. The use of a mixture of pentaneand ether is preferable as this solvent is non-toxic, and may be easilyreplaced by ethanol after extraction of the volatile compounds ofinterest has been achieved.

In some embodiments, the solvent is a mixture of pentane and diethylether, wherein the solvent comprises the pentane and diethyl ether in aweight ratio of pentane to diethyl ether of from about 10:1 to about1:10, such as from about 5:1 to about 1:5, such as from about 3:1 toabout 1:3, such as from 3:1 to 1:1, such as from about 3:1 to about 2:1,such as about 2:1. In some embodiments, the solvent is a mixture ofpentane and diethyl ether, wherein the solvent comprises the pentane anddiethyl ether in a weight ratio of pentane to diethyl ether of fromabout 3:1 to about 1:1. In some embodiments, the solvent is a mixture ofpentane and diethyl ether, wherein the solvent comprises the pentane anddiethyl ether in a weight ratio of pentane to diethyl ether of about2:1.

In some embodiments, the distillation step (ii) and extraction step(iii) (i.e. SDE) are carried out at a pH of no greater than about 2. Insome embodiments, the distillation step (ii) and extraction step (iii)(i.e. SDE) are carried out at a pH of from 0.5 to 2. In someembodiments, the distillation step (ii) and extraction step (iii) (i.e.SDE) are carried out at a pH of from 1 to 2. In some embodiments, thedistillation step (ii) and extraction step (iii) (i.e. SDE) are carriedout at a pH of about 1.6.

It was surprisingly found by the present inventors that, when a pH of nogreater than 2 is used, the amount of volatile compounds extracted fromthe tobacco material is increased. This has the advantage that, whenincorporated into tobacco products (such as smoking/vaping articlesand/or smokeless tobacco products), the flavour obtained from theextract is more pronounced and, although it is still possible toidentify the flavour of tobacco, a series of other desirable notes canalso be perceived as a larger number of different compounds areextracted. It has also been surprisingly found by the present inventorsthat, when the extraction method takes place at pH values of greaterthan 2, towards the end of the extraction process, the pH values tend toincrease thus decreasing the efficiency of the hydrolysis process. Whena pH of about 1.6 is used, even if there is an increase in pH during theSDE process, the end pH value is no greater than 2, thus improving theefficiency of the process for any type of tobacco.

In some embodiments, the pH of no greater than 2 is obtained by mixingthe tobacco material with an acid. In some embodiments, the acid may beselected from hydrochloric acid, sulfuric acid, and mixtures thereof. Insome embodiments, the acid is hydrochloric acid. Treatment withhydrochloric acid (HCl) is the most common hydrolysis method. This isdue to the convenience of application of this reagent, as it can be usedin both the liquid and gas-phase modes. Also, hydrochloric acid findsmany applications in the food industry as it is permitted as a foodacidulent by the Food and Agriculture Organisation of the United Nations(FAO).

In some embodiments, the tobacco material is mixed with the acid (e.g.HCl) at an elevated temperature (such as from about 50° C. to about 80°C.) for no more than about 1 hour before distillation step (ii) andextraction step (iii) (i.e. SDE) are carried out. In some embodiments,the tobacco material is mixed with the acid (e.g. HCl) at an elevatedtemperature (such as from about 50° C. to about 70° C.) for no more thanabout 45 minutes before distillation step (ii) and extraction step (iii)(i.e. SDE) are carried out. In some embodiments, the tobacco material ismixed with the acid (e.g. HCl) at an elevated temperature (such asapproximately 60° C.) for no more than about 30 minutes beforedistillation step (ii) and extraction step (iii) (i.e. SDE) are carriedout. In some embodiments, the tobacco material is mixed with the acid(e.g. HCl) at a temperature of approximately 60° C. for about 20 minutesbefore distillation step (ii) and extraction step (iii) (i.e. SDE) arecarried out.

In some embodiments, the period during which both the distillation step(ii) and the extraction step (iii) (i.e. SDE) are carried out is fromabout 8 to about 15 hours. In some embodiments, the period during whichboth the distillation step (ii) and the extraction step (iii) (i.e. SDE)are carried out is from about 8 to about 10 hours. In some embodiments,the period during which both the distillation step (ii) and theextraction step (iii) (i.e. SDE) are carried out is about 9 hours. Insome embodiments, the period during which both the distillation step(ii) and the extraction step (iii) (i.e. SDE) are carried out is about 8hours.

As the skilled person will appreciate, the typical time for SDE to becarried out is around 5 hours. It was surprisingly found by the presentinventors that, when the SDE is carried for a period of at least 8 hours(and preferably comprising only a single step in the SDE process forthis period of at least 8 hours), the concentration of the volatilecompounds in the resulting extract (or distillate) was increasedsignificantly. This has the advantage that, when incorporated intotobacco products (such as smoking/vaping articles and/or smokelesstobacco products), the sensory characteristics of the tobacco productsare improved and the flavour obtained from the extract is morepronounced.

In some embodiments, the distillation step (ii) and the extraction step(iii) (i.e. SDE) are carried out at a temperature of up to about 130° C.

In some embodiments, the distillation step (ii) is carried out at atemperature of from about 100° C. to about 130° C., such as from about105° C. to about 125° C., such as from about 110° C. to about 125° C.,such as from about 115° C. to about 125° C., such as from about 120° C.to about 122° C., such as approximately 121° C.

In some embodiments, the extraction step (iii) is carried out at atemperature of from about 50° C. to about 100° C., such as from about60° C. to about 95° C., such as from about 70° C. to about 90° C., suchas from about 75° C. to about 90° C., such as from about 80° C. to about90° C., such as from about 85° C. to about 90° C.

In some embodiments, the distillation step (ii) and the extraction step(iii) (i.e. SDE) are carried out at atmospheric pressure.

In some embodiments, the distillation step (ii) and extraction step(iii) are carried out simultaneously in one single step. As such, insome embodiments, the SDE method described herein is carried out in justone single step. In some embodiments, an additional distillation and/orextraction step is excluded. In some embodiments, the entire SDE method(i.e. distillation step (ii) and extraction step (iii)) is carried outsimultaneously in one single piece of apparatus with no additionalprocessing prior to removal of the extracted product from the apparatus.

In some embodiments, the one or more volatile compounds of interestextracted from the tobacco material are selected from the groupconsisting of: sugars, sugar esters, amino acids, beta-carotene,violaxanthin, lutein, neoxanthin, phytol, labdanoids, cembranoids,polyphenols, lignin, and mixtures thereof.

In some embodiments, the one or more volatile compounds of interestextracted from the tobacco material are selected from the groupconsisting of: methyl butanol, benzyl alcohol, phenylethanol,methoxyvinylphenol, vinylphenol, hydroxydamascone, furfural,hydroxymethylfurfural, furaneol, cyclotene, dimethylpyrazines,trimethylpyrazine, tetramethylpyrazine, hydroxymethyl(methyl)pyrazines,isophorone, ketoisophorone, safranal, iononas, damascenone,beta-damascenone, megastigmatrienone, beta-damascone, beta-ionone,2,6-nonadienal, 2-nonenal, linalool, linalool oxide, geranyl acetone,farnesyl acetone, methylheptadienone, solanone, solanascone,norambrenolide, ambroxide, sclareolide, isobutyric acid, isovalericacid, 3-methylvaleric acid, heptanoic acid, benzoic acid, phenylaceticacid, ortho, para-cresol, methional, guaiacol, vinylphenol,ethylguaicol, vinylguaiacol, eugenol, vanillin and mixtures thereof.

In some embodiments, the one or more volatile compounds of interestextracted from the tobacco material are responsible for a characteristictaste or flavour of at least one tobacco variety.

In some embodiments, the extract obtained from the tobacco material issubstantially free of alkaloids and/or tobacco-specific nitrosamines(TSNAs). In some embodiments, the extract obtained from the tobaccomaterial is substantially free of nicotine and/or TSNAs.

As used herein “substantially free of alkaloids” means that the extractcomprises less than about 15% by weight of alkaloids (such as nicotine),such as less than about 10% by weight of alkaloids (such as nicotine),such as less than about 5% by weight of alkaloids (such as nicotine),such as less than about 2% by weight of alkaloids (such as nicotine),such as less than about 1% by weight of alkaloids (such as nicotine),such as less than about 0.5% by weight of alkaloids (such as nicotine),such as less than about 0.1% by weight of alkaloids (such as nicotine).In some embodiments, the extract obtained from the tobacco materialcomprises less than about 1% by weight of alkaloids (such as nicotine).

Without wishing to be bound by theory, the use of a pH of no greaterthan 2 allows any nicotine present in the tobacco material to react withhydrochloric acid (HCl) to form a water-soluble salt, nicotinehydrochloride. This salt is not extracted during the method of thepresent invention.

As used herein “substantially free of TSNAs” means that the extractcomprises less than about 15% by weight of TSNAs, such as less thanabout 10% by weight of TSNAs, such as less than about 5% by weight ofTSNAs, such as less than about 2% by weight of TSNAs, such as less thanabout 1% by weight of TSNAs, such as less than about 0.5% by weight ofTSNAs, such as less than about 0.1% by weight of TSNAs. In someembodiments, the extract obtained from the tobacco material comprisesless than about 1% by weight of TSNAs.

In some embodiments, further processing of the extracted product may becarried out after the distillation step (ii) and the extraction step(iii) (i.e. SDE) have been carried out. It is noted that this furtherprocessing is not considered to be an additional step in the SDE methoditself, but rather an additional processing step on the distillateobtained from the SDE method.

The method of further processing may be carried out in several ways. Themethod of further processing may depend on the compounds present in theextract and/or the type of solvent used in the extraction. For example,in some embodiments, when an organic non-polar solvent (e.g., pentane,diethyl ether or a mixture thereof) is used to extract one or morecompounds from the steam distilled tobacco material, the solvent, afterbeing in contact with the distilled material, may simply be filtered ordried to remove particulate tobacco material and the solvent, and thefiltrate may be concentrated.

In some embodiments, further processing of the extracted product iscarried out, the further processing comprising a purifying step whichcomprises replacing the solvent used for extraction with another solventsuitable for use in a tobacco product. In some embodiments, such apurifying step comprises replacing the solvent used for extraction(e.g., pentane, diethyl ether or a mixture thereof, preferably a 2:1mixture of pentane:diethyl ether) with ethanol. The replacement of thesolvent with ethanol is desirable for use in tobacco products.

In some embodiments, further processing of the extracted product iscarried out, the further processing comprising subjecting an isolatedcompound or mixtures of these compounds to conditions that leadthis/those compound/s to suffer chemical transformation. For example,the tobacco material obtained from plants of the Nicotiana species or aportion of these plants and also the extracts containing the isolatedcompound(s) may be treated to cause chemical transformation and/or beadmixed with other ingredients. The chemical transformations ormodification of the tobacco material, extract, or isolated compound(s)can result in changes of certain chemical and physical properties of thetobacco material, extract, or isolated compound(s) (e.g., the sensoryattributes thereof). Exemplary chemical modification processes includeacid/base reaction, hydrolysis, oxidation, heating and/or enzymatictreatments. As a result of these processes, compounds can undergovarious degradation reactions.

In some embodiments, the one or more volatile compounds of interestextracted from the tobacco material are degradation products selectedfrom the group consisting of: sugars, sugar esters, amino acids,beta-carotene, violaxanthin, lutein, neoxanthin, phytol, labdanoids,cembranoids, polyphenols and lignin, and mixtures thereof. In someembodiments, such degradation products may be further treated to providevarious flavour compounds, selected from the group consisting ofmethylbutanol, benzyl alcohol, phenylethanol, methoxyvinylphenol,vinylphenol, hydroxydamascone, furfural, hydroxymethylfurfural,furaneol, cyclotene, dimethylpyrazines, trimethylpyrazine,tetramethylpyrazine, hydroxymethyl(methyl)pyrazines, isophorone,ketoisophorone, safranal, iononas, beta-damascenone, megastigmatrienone,beta-damascone, beta-ionone, 2,6-nonadienal, 2-nonenal, linalool,linalool oxide, geranyl acetone, farnesyl acetone, methylheptadienone,solanone, solanascone, norambrenolide, ambroxide, sclareolide,isobutyric acid, isovaleric acid, 3-methylvaleric acid, heptanoic acid,benzoic acid, phenylacetic acid, ortho-cresol, para-cresol, methional,guaiacol, vinylphenol, ethylguaicol, vinylguaiacol, eugenol, andvanillin.

As used herein, “degradation products” are any compounds that areproduced from the compounds extracted and/or degraded according to thepresent invention. Degradation products can be formed naturally fromsuch compounds or may be produced by an accelerated degradation process(e.g., by the addition of heat and/or chemicals to accelerate thebreakdown of the compounds). These compounds can be degraded, forexample, by means of oxidation and/or hydrolysis reactions (e.g.,through treatment with hydrochloric acid or other acid agent).

In some embodiments, residual tobacco material is present in the wastematerial resulting from the extraction method described herein. In someembodiments, the waste material resulting from the extraction method asdescribed herein may be used as a raw material for reconstituted tobaccoor briquette production.

Tobacco Extract

A second aspect of the invention provides a tobacco extract obtainableor obtained by the method as described above, wherein the tobaccoextract comprises one or more volatile compounds of interest in anamount of at least about 75% by weight of the tobacco extract.

In some embodiments, the tobacco extract is obtainable or obtained bythe method comprising the steps of:

-   -   i) providing tobacco material;    -   ii) subjecting the tobacco material to steam distillation; and    -   iii) extracting one or more volatile compounds of interest from        the tobacco material with a solvent;        wherein distillation step (ii) and extraction step (iii) are        carried out simultaneously and at a pH of no greater than 2, and        wherein the period during which both the distillation step (ii)        and the extraction step (iii) are carried out is from about 8 to        about 20 hours; wherein the tobacco extract comprises one or        more volatile compounds of interest in an amount of at least        about 75% by weight of the tobacco extract.

In some embodiments, the tobacco extract is obtainable or obtained by amethod as defined in any one of the embodiments described above.

In some embodiments, the tobacco extract comprises the one or morevolatile compounds of interest in an amount of at least about 80% byweight of the tobacco extract, such as in an amount of at least about85% by weight of the tobacco extract, such as in an amount of at leastabout 90% by weight of the tobacco extract, such as in an amount of atleast about 95% by weight of the tobacco extract, such as in an amountof at least about 97% by weight of the tobacco extract.

In some embodiments, the tobacco extract comprises the one or morevolatile compounds of interest in an amount of at least about 95% byweight of the tobacco extract.

In some embodiments, the one or more volatile compounds of interest inthe tobacco extract are selected from the group consisting of: sugars,sugar esters, amino acids, beta-carotene, violaxanthin, lutein,neoxanthin, phytol, labdanoids, cembranoids, polyphenols, lignin, andmixtures thereof.

In some embodiments, the one or more volatile compounds of interest inthe tobacco extract are selected from the group consisting of: methylbutanol, benzyl alcohol, phenylethanol, methoxyvinylphenol, vinylphenol,hydroxydamascone, furfural, hydroxymethylfurfural, furaneol, cyclotene,dimethylpyrazines, trimethylpyrazine, tetramethylpyrazine,hydroxymethyl(methyl)pyrazines, isophorone, ketoisophorone, safranal,iononas, damascenone, beta-damascenone, megastigmatrienone,beta-damascone, beta-ionone, 2,6-nonadienal, 2-nonenal, linalool,linalool oxide, geranyl acetone, farnesyl acetone, methylheptadienone,solanone, solanascone, norambrenolide, ambroxide, sclareolide,isobutyric acid, isovaleric acid, 3-methylvaleric acid, heptanoic acid,benzoic acid, phenylacetic acid, ortho, para-cresol, methional,guaiacol, vinylphenol, ethylguaicol, vinylguaiacol, eugenol, vanillinand mixtures thereof.

In some embodiments, the tobacco extract is substantially free ofalkaloids and/or tobacco-specific nitrosamines (TSNAs). In someembodiments, the tobacco extract is substantially free of nicotineand/or TNSAs.

As used herein “substantially free of alkaloids” means that the tobaccoextract comprises less than about 15% by weight of alkaloids (such asnicotine), such as less than about 10% by weight of alkaloids (such asnicotine), such as less than about 5% by weight of alkaloids (such asnicotine), such as less than about 2% by weight of alkaloids (such asnicotine), such as less than about 1% by weight of alkaloids (such asnicotine), such as less than about 0.5% by weight of alkaloids (such asnicotine), such as less than about 0.1% by weight of alkaloids (such asnicotine). In some embodiments, the tobacco extract comprises less thanabout 1% by weight of alkaloids (such as nicotine).

As used herein “substantially free of TSNAs” means that the tobaccoextract comprises less than about 15% by weight of TSNAs, such as lessthan about 10% by weight of TSNAs, such as less than about 5% by weightof TSNAs, such as less than about 2% by weight of TSNAs, such as lessthan about 1% by weight of TSNAs, such as less than about 0.5% by weightof TSNAs, such as less than about 0.1% by weight of TSNAs. In someembodiments, the tobacco extract comprises less than about 1% by weightof TSNAs.

Tobacco Product

A third aspect of the invention provides a tobacco product comprising atobacco extract as defined above.

In some embodiments, the tobacco product is a smoking/vaping article ora smokeless tobacco product, an electronic device, a heating device, ora hybrid of both, a cigarette, a cigar, or a smokeless oral tobaccoproduct.

In some embodiments, the tobacco product is a smoking/vaping article ora smokeless tobacco product. In some embodiments, the tobacco product isa cigarette, a cigar, or a smokeless oral tobacco product.

The tobacco extract may be employed as a component of a tobacco productin a variety of ways. The tobacco extract may be employed as a componentof processed tobaccos. In some embodiments, the tobacco extract may beemployed within a flavour or casing formulation for application to atobacco strip or within a top dressing formulation. Alternatively, thetobacco extract may be employed as an ingredient of a reconstitutedtobacco material. The tobacco extract may be incorporated into acigarette filter (e.g., in the filter plug, plug wrap, or tipping paper)or incorporated into cigarette wrapping paper, preferably on the insidesurface, during the cigarette manufacturing process.

In some embodiments, the tobacco extract is included in the tobaccoproduct in the form of a flavourant, a casing or a combination thereof,or in the form of reconstituted tobacco which includes the tobaccoextract.

In some embodiments, the tobacco product is an electronic device, aheating device, or a hybrid of both. In such embodiments, theapplication of the tobacco extract may be adapted to the respectivecharacteristics of those technologies. For example, in some embodiments,the tobacco product is an electronic device (e.g. an electroniccigarette) comprising the tobacco extract in the inhalable liquidcontained therein. In some embodiments, the tobacco product is a heatingdevice comprising the tobacco extract in the tobacco, filter orconsumable paper included therein.

The amount of tobacco product may comprise the tobacco extract in anysuitable amount depending on the desired function of the tobaccoextract, the chemical composition of the extract and the type of tobaccoproduct to which the extract is added. In some embodiments, the tobaccoproduct comprises the tobacco extract in an amount of from about 0.0001%to about 15% of the tobacco product based on the total dry weight of thetobacco product to which the extract is added. In some embodiments, thetobacco product comprises the tobacco extract in an amount of from about0.01% to about 10% by weight of the tobacco product based on the totaldry weight of the tobacco product to which the extract is added. In someembodiments, the tobacco product comprises the tobacco extract in anamount of from about 0.1% to about 5% by weight of the tobacco productbased on the total dry weight of the tobacco product to which theextract is added.

Method of Preparing Tobacco Product

A fourth aspect of the invention provides a method of preparing thetobacco product described herein, the method comprising the steps of:

-   -   (a) preparing a tobacco extract in accordance with a method as        described herein; and    -   (b) combining the tobacco extract directly with a tobacco        product and/or combining the tobacco extract with reconstituted        tobacco and optionally combining the reconstituted tobacco with        a tobacco product.

In some embodiments the method of preparing the tobacco productcomprises the steps of:

-   -   (a) preparing a tobacco extract in accordance with a method as        described herein; and    -   (b) combining the tobacco extract directly with a tobacco        product.

In some embodiments, the tobacco extract is combined directly with atobacco product by adding the tobacco extract to the tobacco product inits liquid form, by adding the tobacco extract to the tobacco product inthe form of a casing, or by a combination thereof.

In some embodiments the method of preparing the tobacco productcomprises the steps of:

-   -   (a) preparing a tobacco extract in accordance with a method as        described herein; and    -   (b) combining the tobacco extract with reconstituted tobacco,        and combining the reconstituted tobacco with a tobacco product.        Use

A fifth aspect of the invention provides the use of the tobacco extractas described herein for improving the sensory properties of a tobaccoproduct.

In some embodiments, there is provided the use of a tobacco extractobtained or obtainable from the method described herein for improvingthe sensory properties of a tobacco product.

In some embodiments, the tobacco extract described herein is used forenhancing the flavour of a tobacco product.

EXAMPLES Example 1

The method according to the invention was carried out using theapparatus shown in FIG. 3 . The apparatus shown in FIG. 3 is usefulwhere the solvent used is less dense than water (such as pentane:diethylether (2:1)). As shown in FIG. 3 , the apparatus 20 includes a 100 mLflask 21 for the tobacco sample, a 2 mL flask 22 for the solvent, waterbaths 23 and a heating/stirring plate 24 for heating the samples. Theapparatus 20 also includes an auxiliary heating coil 25 for heating thetobacco sample to a higher temperature than the solvent such that steamdistillation may be carried out. During the process, vapours condensedon the cold finger 27, and the extraction process began between bothliquid films on the condenser surface 28. Each of tubes 26 a, 26 b havean internal diameter of about 4 mm.

To the 2 mL flask 22 of the apparatus shown in FIG. 3 , 1 mL ofpentane:diethyl ether (2:1) mixture and fragments of pumice stone wereadded. In the 100 mL flask 21, 5 g of tobacco (dry bases weight; variousgrades of Burley and Virginia tobacco were used as the tobacco material,some considered of high and others of low quality), fragments of pumicestone, bi-distilled water and about 1 mL of hydrochloric acid (HCl 37%)were added such that the pH desired for the aqueous solution in contactwith the tobacco was attained and the volume of this solution remainedequal to 40 mL. Preliminary tests showed that the tobacco should be leftin contact with the acid for 20 minutes, at about 60° C., to assure thatthe pH desired will be maintained in the SDE system after mixing theacid solution with the tobacco.

The two flasks 21 and 22 were then connected to the body of theapparatus and the bi-distilled water and the pentane:diethyl ether (2:1)were mixed until the two liquids started returning to their respectiveflasks.

The flasks 21 and 22 were placed in baths previously heated up to 100and 140° C., respectively, such that the levels in the flasks remainedslightly above the levels of the baths. When the liquids in the flasksbegan to boil, it was observed whether the temperature in the smallerbath stabilised between 85 and 90° C. and that of the bigger flaskbetween 120 and 122° C. Adjustments were made, where necessary, in orderthat the SDE process is accomplished in the recommended temperatureranges.

The system was operated for 9 hours and, during this period, it wasobserved whether the level in the flask containing the extract wasconstant. A little more of the pentane:diethyl ether (2:1) mixture wasadded, when necessary, through the system's side-outlet in order toensure that the level of liquid in the flask containing the extract waskept constant.

After 9 hours, the equipment was lifted out of the water baths and leftto cool until the extract temperature equilibrated to room temperature.The extract was transferred to a calibrated tube and the volume adjustedto 1.5 mL with the pentane:diethyl ether (2:1) mixture.

Exchange of Pentane:Diethyl Ether Solution for Ethanol

1.5 mL of absolute ethanol and fragments of pumice stone were added tothe tube containing the extract. The solution was concentrated up toapproximately 1.5 mL in a bath at 60° C. When the boiling ceased, thetube was kept in contact with the bath for 30 minutes more to ensurethat both the pentane and diethyl ether had been removed. Thepentane:diethyl ether solvent had thus been replaced by ethanol solvent,and the extract was now in ethanol solution. The extract in ethanolcould be injected directly into cigarettes, applied to tobacco beforecigarette preparation or applied in electronic, heating or hybriddevices for a subsequent sensorial evaluation.

It was found that the addition of 1 μl of the extract to a cigarette wascapable of clearly altering the organoleptic characteristics of thesmoke of the tobacco product, and even 0.5 μl was considered to be anacceptable level of addition.

Considering that this level of addition corresponds to the applicationof the extract obtained from 6 kg of tobacco over 3 tons of the tobaccoto be used for cigarette manufacturing, it can be concluded that theextracts are highly potent. The same can be said regarding theirstability, since the extract was found to be able to be stored for sixmonths without having its characteristics altered.

It was also found that extracts obtained by SDE in acid medium presenteda great potential as flavours for cigarettes due to the characteristicswhich they impart to the smoke. Also, a decrease in irritatingsensations, an increase in the amplitude and the fact that they enhancethe tobacco basic notes, such as green and earthy, without impartingartificial characteristics to the cigarettes was further noted.Moreover, the extracts were found not to present any problems from atoxicological point of view to be used as flavours for cigarettes.

The same approach detailed above was conducted to evaluate the extracttaste performance into capsules and tobacco products such as electronicdevices, heating devices and hybrid devices, and the potential for useof the extracts in such products was confirmed.

Example 2

An apparatus 30 as shown in FIG. 4 was used in order to extract volatilecompounds of interest from a larger sample of tobacco material. In thisExample, 2240 g of tobacco, 933 mL of hydrochloric acid 6 N (466.5 mL ofHCl 37%+466.5 mL of water), 15 L of water and 100 g of pumice stones(PA), were placed in a 24 L round bottom flask 31 on a heating mantle.The mixture was stirred carefully with the aid of a glass rod in orderto obtain a homogeneous mixture. 200 mL of pentane:diethyl ether (2:1)mixture with 5 g of pumice stones was placed in the smaller round bottomflask 32.

The body of the SDE apparatus was connected to the flasks with the aidof claws so that the system became free of tension. After connecting theflasks to the apparatus, some drops of water were placed (with the aidof a pipette) in the joints to assist in the sealing. With the aid of apipette, water was added to the central part of the apparatus body toraise the level, on the left side arm, to near the return level.

With the aid of a pipette, the pentane:diethyl ether (2:1) mixture wasslowly added to the body of the apparatus through the walls until thelevel rise on the right side arm at the level of return. In case thereturn levels were not achieved at the same time with the systemvertically, the apparatus was tilted slightly as required.

The condensers were connected to the body of the apparatus and thecooling system turned on (commercial ethanol:water—2:1).

Both round bottom flasks were heated. This heating was achieved byconnecting the voltage controllers that were connected to the heatingmantles where the two flasks were situated on the positions previouslydetermined. These positions enabled the temperatures to be sufficientfor the liquids contained in the two flasks to come to the boil withoutturbulence.

When the solution in the smaller flask began to reflux, the beginning ofthe 9 h of distillation was started. Throughout this period, the largestflask and the side arms of the apparatus were kept covered with asbestosor aluminium foil.

Until the system reached the equilibrium (the two liquids simultaneouslydistilling and returning to their flasks), the system required continuedattention. If one of the solvent layers (pentane-ether or water) raisedmore than should, tending to move to the opposite flask, the system hadto be tilted slightly to the side of the solvent which was in excess.

In case the volume of pentane-ether decreased during the SDE process,more was added with the aid of a pipette by the top of the condenser,thus restoring the lost amount of solution.

After the period of 9 hours of distillation, the heating was turned offand the system left until boiling had ceased. After the boiling hadceased, the flask containing the pentane-ether was removed and 45 mL ofabsolute ethanol was added to this mixture. The solution wasconcentrated in an apparatus for distillation at 45° C. until no furtherboiling was observed. The temperature was raised to 65° C. for 15 moreminutes. After these 15 minutes, the heating was turned off, and thesystem left to cool until the solution reached room temperature. Thesolution was then transferred to a beaker and the volume made up to 45mL with absolute ethanol. This final solution was then ready to be usedas flavouring.

It is noted that, the procedure described above was carried out on anapparatus as shown in FIG. 4 . It was found that, carrying out theprocedure on an apparatus 40 as shown in FIG. 5 (which is merely anenlargement of the apparatus used for 5 g of tobacco in Example 1, andwhich is shown in FIG. 3 ), resulted in slight difficulties. It was, forinstance, difficult for the distilled pentane and ethyl ether to returnto the equipment's body due to the great amount of water that enteredthe system below them.

Therefore, the apparatus shown in FIG. 4 was designed, wherein the twocondensers at the top part of the equipment shown in FIG. 5 were adaptedto allow the distillation of the water, at an acceptable rate, withoutlosing the more volatile compounds. The apparatus shown in FIG. 4 wasdesigned to by-pass the above-identified problems when obtainingextracts from 2240 g of tobacco. As shown in FIG. 4 , the watercontaining the compounds distilled and the organic solvents werecondensed in independent condensers and only met each other when theywere already cold. This made it possible to carry out the heating up ofthe tobacco with the acid solution in the desired way, withoutinterfering in the return of the distilled ethyl ether and pentane.

Comparative Example 1—Comparison of SDE Method with Neutral VolatileScan (NVS)

A comparison of the chromatograms of extracts obtained by SDE and NVSwas made.

Through the NVS method, it was postulated that the compounds naturallyfound in tobacco are isolated, and practically all the substancespresent in the SDE extract should be present in the NVS extract, if theprocess which led to their isolation were a simple steam distillation.

In order to carry out a comparison between the extracts, several Burleyand Virginia tobacco grades were evaluated.

The procedure for tobacco analysis by the NVS method was as follows:

-   -   1. To a Soxhlet extractor (500 ml flask, Whatman 33×80 mm        cellulose cartridge), 300 ml of dichloromethane and 5.0 g of        tobacco were added. The flask was heated in a water bath at        50-55° C. for 6.5 hours. Extraction time could be lengthened to        16 hours, if considered suitable. Throughout the extraction        period, the system was maintained in an inert atmosphere and        exposing the extract to the light was avoided by covering the        lower part of the apparatus with aluminium foil. The extract was        transferred into a 500 ml separation funnel and the flask washed        with small portions of dichloromethane. More dichloromethane was        added to the extract in cases where the volume was less than 300        ml.        -   To create an inert atmosphere in the apparatus used for            tobacco extraction and in the apparatuses identified below            in steps 4 and 6, a flexible tube was used to couple the            apparatus exit with a 2.0 cm I.D. glass tube through which            nitrogen gas is passed at an approximate flow rate of 100            ml/min. Prior to system warm-up, the glass tube end was            closed, and the apparatus joints maintained half-open so as            to force nitrogen gas to run through the apparatus. After            fifteen minutes, the glass tube end was opened, and the            joints properly adjusted. To prevent the solvent not            condensed during extractions or distillations from            accumulating on the flexible tube (thus running back into            the apparatus and then contaminating it), the glass tube was            maintained below the point at which it is coupled with the            apparatus, a flexible tube with proper internal diameter was            used, and the apparatus modified (if need be) so that the            exit onto which the flexbile tube is coupled is in a            downslope condition.    -   2. The extract was then partitioned six times with 150 ml of a        pH=6.0 buffer solution containing 0.092M Na₂HPO₄ and 0.48M        KH₂PO₄. The solutions were stirred for 15 seconds and rested for        5 minutes prior to discarding the aqueous phase. If the        separation of the two phases was not clear at the interface,        most of the organic phase was removed from the funnel, 10 ml of        dichloromethane added thereto, and stirred again and allowed to        rest until the separation between the phases was complete.    -   3. After partitioning, the organic phase was run through a        2.5×200 mm glass column containing 50 g anhydrous sodium        sulfate. Column flow rate was approximately 5 ml/min. The column        was washed with dichloromethane.    -   4. The solution was concentrated in an apparatus as described        above in step 1. The flask was heated in a water bath at 55-60°        C., and the entire system maintained in an inert atmosphere as        described above in step 1, and its lower part prevented from        light exposure by covering it with an aluminium foil. The        extract volume was reduced to approximately 1 ml. The residue        was transferred into a graduated tube and made up to a volume of        4 ml with a 7.5% v/v ethanol solution in dichloromethane.    -   5. To a 15 cm×15 mm I.D. glass column was added 1.75 g 100-200        mesh Florisil together with a 7.5% v/v solution of ethanol in        dichloromethane. The excess solution was removed until its level        was just above that of the Florisil, and the extract obtained in        step 4 was applied thereto. The resultant mixture was eluted        with a solution identical with that used in column preparation,        and 40 ml collected therefrom.    -   6. 40 ml of dichloromethane was added to the solution collected,        and the solution was concentrated to a volume of approximately 1        ml, as described above in step 4. 60 μl of ethyl palmitate        (internal standard) solution in ethanol was added, homogenised        by manual stirring, and the concentrate transferred to a        graduated tube. The solution was made up to a volume of 3 ml        with dichloromethane.    -   7. The resulting extract was then analysed by gas chromatography        using a Hewlett-Packard chromatograph, model 5880A, under the        following conditions:        -   Carrier gas: H₂, 20 psi; t_(M) 120° C., 0.70 min; split            ratio 15:1.        -   Injected volume: 2 μl.        -   Injection technique: fill syringe with solvent prior to            pulling in the extract and inject with hot-needle technique,            as described in Grob, Jr, K. & Neurom, H. P., J. High Resol.            Chromat. Chromat. Comm., 15-21, 1979.        -   Integrator: Hewlett-Packard 3356B Laboratory Automation            System.        -   Column: 25 m×0.2 mm I.D., crosslinked methyl silicone; film            thickness 0.50 μm, fused silica.        -   Oven temperature profile: initial value—120° C.; initial            time—0.00 min            -   Level 1:                -   Program rate: 1.50° C./min                -   Final value: 155° C.                -   Final time: 0.00 min            -   Level 2:                -   Program rate 0.80° C./min                -   Final value: 210° C.                -   Final time: 0.00 min            -   Level 3 (column purge time):                -   Program rate: 30.00° C./min                -   Final value: 300° C.                -   Final time: 35 min        -   Detector: FID; 300° C.; H2, 30 ml/min; air, 300 ml/min;            nitrogen/makeup, 30 ml/min        -   Injector: 200° C.; mixing chamber type liner        -   Additional commands (actuated automatically)            -   90.00 min—signal off (recorder)            -   90.00 min—injector temperature, 275° C.            -   115.00 min—injector temperature, 200° C.

The SDE method was carried out using the method described in Example 1,except that a pH of 5.0-6.0 was used, which is not in accordance withthe present invention.

Table 1 shows the results obtained in one of these comparisons, whichconfirms the above conclusions.

TABLE 1 comparison of the relative areas of various peaks of flavourcompounds isolated by the NVS and SDE methods from Virginia tobacco NVSSDE (relative area of (relative area of Compound chromatogram peaks)chromatogram peaks) Solanone 13.4 69.4 Damascenone 1.0 3.1 Solanol 1.39.8 beta-Damascone 1.0 6.8 Megastigmatrienone* 86.0 258.8 Solanascone1.0 32.7 *Total of the megastigmatrienone isomers

It can be seen that many of the substances in the extract obtained bySDE were either present in a smaller amount or were not present at allin the NVS extract.

Compounds shown in Table 1, such as megastigmatrienone, solanone anddamascenone, are very important flavours and their presence in largeramounts in the SDE extract justifies the differences in flavour producedby the SDE extract as compared with that produced by the NVS extract.

Upon application of each of the extracts to tobacco products, it wasfound that the flavour of the extracts isolated from the tobacco throughthe extraction with solvents (i.e. NVS) is rather less pronounced thanfor the extracts isolated through the use of the SDE method describedherein.

Based on these results, and without wishing to be bound by theory, itseems that the following processes may be occurring in the treatment ofthe tobacco in a SDE system: simple distillation, steam distillation andalso degradation/steam distillation or degradation/simple distillation.Since the pH of the aqueous solution in contact with the tobacco duringSDE is in the range of 5.0 to 6.0 and since the tobacco is heated up forsome hours at about 100° C., degrading processes such as hydrolysis,dehydration, isomerization and simple thermal degradation may behappening. These processes, when occurring in an SDE system, maygenerate products with characteristics distinct from the ones obtainedin closed systems.

Without wishing to be bound by theory, in closed systems, for example,the tendency is to obtain the most stable products and volatilesubstances naturally found in tobacco, or generated from it, have asmany chances of undergoing chemical reactions as the least volatileones. In SDE systems, volatile substances have fewer chances of beingtransformed because they are quickly removed. Therefore, relativelyunstable compounds, or even intermediates of degrading processes, may beisolated.

Comparative Example 2—Comparison of SDE Carried Out at pH 6.0 and pH 2.0

In order to demonstrate the advantages of using a pH of no greater than2 in the SDE method, the extracts obtained from SDE using a pH value of2.0 and a pH value of 6.0 were evaluated and compared.

The method of Example 1 was carried out using Virginia tobacco as thetobacco material at a pH of 2.0 (as described above) and also at a pH of6.0 by using different amounts of HCl (e.g., 1 mL of HCl 37% to achievepH lower than 2) in order to vary the pH of the procedure. The extractsthus obtained were separated by gas chromatography and theirchromatograms compared.

For reference, an exemplary chromatogram is shown in FIG. 6 , whichseparates the peaks of the chromatogram into three groups: volatileflavours, semi-volatile flavours and diterpenes. It is noted that FIG. 6is for reference only, and is to be used for interpretation of the peaksof the chromatograms shown herein.

The chromatograms obtained from both SDE methods at pH=2.0 and pH=6.0are shown in FIG. 7(a) and FIG. 7(b), respectively. As can be seen inFIG. 7 , at a pH of no greater than 2, the formation of volatilecompounds is rendered highly propitious as the concentration of thevolatile compounds extracted at pH is significantly increased. In termsof flavour, the changes are also quite pronounced. Comparing the twoextracts, it is noticed that the flavour obtained at the pH of nogreater than 2 is more pronounced and, although it is still possible toidentify the flavour of tobacco, a series of other notes can also beperceived.

TABLE 2 comparison of the relative areas of various peaks of flavourcompounds isolated by the SDE method at pH = 6.0 and pH = 2.0 fromVirginia tobacco Compound pH 6.0 pH 2.0 Solanone 83.3 191.8 Damascenone7.3 8.7 Norsolonadione 3.0 8.7 Megastigmatrienone* 200.3 417.3Solanascone 112.9 146.1 *Total of the megastigmatrienone isomers

The relative areas of various peaks present in the extracts obtained bySDE at pH 6.0 and pH 2.0 can be seen in Table 2, where several flavoursrecognized as important are shown, many of which are utilized informulations for aromatizing tobacco. All of the flavour compounds havehad their peaks increased (i.e. their concentration was higher) by thetreatment at the lower pH of 2. In addition, many other compounds whosestructures have not been determined as yet were also extracted in largerconcentrations at pH 2.0.

In addition, a further experiment was carried out to determine theeffect of carrying out the SDE method at pH 2.0 when the tobaccomaterial is mixed with the acid several hours before SDE as comparedwith treating the tobacco material with acid immediately prior to SDE.

In this further experiment, the tobacco was treated with an acidsolution for several hours, at about 90° C., without removing thevolatile substances. Then, the acid was neutralized and the SDE carriedout. The extract thus obtained presented a chemical compositiondifferent from the one obtained by submitting the tobacco to a treatmentwith the acid solution, simultaneously with the SDE. As an illustration,the solanone content, which is one of the compounds that have theirformation increased with the treatment by SDE in acid medium, decreasedin the extract obtained from the tobacco which had been treated withacid well in advance of SDE, and submitted to the SDE separately.

Comparative Example 3—Comparison of SDE Carried Out at a Range ofDifferent pH Values

In order to further demonstrate the advantages of using a pH of nogreater than 2 in the SDE method, the extracts obtained from SDE using arange of pH values were evaluated and compared.

The method of Example 1 was carried out for a range of samples eachusing Virginia tobacco as the tobacco material in each sample, and atdifferent pH values by varying the amount of HCl used in order to varythe pH of the procedure. Extracts obtained by SDE at pH values of 6.0,4.0, 3.0, 2.0 and 0.5 were evaluated. The extracts thus obtained wereseparated by gas chromatography and their chromatograms compared.

The chromatograms obtained from the SDE methods at each of the differentpH values are shown in FIGS. 8-12 .

As can be seen from a comparison of each of FIGS. 8 to 12 , theformation of volatile compounds is significantly increased at pH valuesof no greater than 2.

The extracts obtained were also tested as flavours for cigarettes. Forthe extracts produced using SDE at a pH of 2 or 0.5, among thesensations imparted by them to the smoke, even at very low levels ofaddition, are increase in amplitude, the diminishing of irritantsensations and the increase of tobacco characteristic notes according totobacco variety used for extract production. Based on these results, itcan be concluded that tobacco extracts obtained by SDE in acidconditions have the chance of becoming important from commercial pointof view, making it possible, for example, the improvement of low qualitytobaccos, increase the aroma of low tar cigarettes and even be used innew technologies like capsules or even in next generation products as atobacco taste enhancer or simply to mask the undesired sensoryattributes of such products.

Comparative Example 4—Comparison of SDE Carried Out for DifferentPeriods of Time

In order to demonstrate the advantages of carrying out the SDE methodfor a period of from about 8 to about 20 hours, the extracts obtainedfrom SDE over a range of time periods were evaluated and compared.

The SDE method described in Example 1 was performed, but for a totalperiod of 5 hours rather than the 9 hours of Example 1. As the skilledperson will appreciate, 5 hours is generally regarded as being thestandardized period of time for the extraction in the SDE method for theanalysis of the tobacco.

It was found by the present inventors that this period of 5 hours wasnot sufficient for the quantitative removal of the volatile compounds.In contrast, the SDE carried out for a period of 9 hours producedflavour compounds in higher concentrations, thus providing enhancedflavour. The results obtained in this study have indicated that theperiod of time of SDE in acid medium significantly influences theextracts chemical composition.

Regarding the importance in organoleptic terms of the compounds isolatedafter a long period of time (i.e. at least about 8 hours) of SDE in acidmedium, it was found that at least some of them are capable ofcontributing to the smoke/vaping quality.

The various embodiments described herein are presented only to assist inunderstanding and teaching the claimed features. These embodiments areprovided as a representative sample of embodiments only, and are notexhaustive and/or exclusive. It is to be understood that advantages,embodiments, examples, functions, features, structures and/or otheraspects described herein are not to be considered limitations on thescope of the invention as defined by the claims or limitations onequivalents to the claims, and that other embodiments may be utilisedand modifications may be made without departing from the scope of theclaimed invention. Various embodiments of the invention may suitablycomprise, consist of, or consist essentially of appropriate combinationsof the disclosed elements, components, features, parts, steps, means,etc., other than those specifically described herein. In addition, thisdisclosure may include other inventions not presently claimed, but whichmay be claimed in the future.

The invention claimed is:
 1. A method of extracting one or more volatilecompounds of interest from tobacco material, the method comprising thesteps of: i) providing tobacco material; ii) subjecting the tobaccomaterial to steam distillation; and iii) extracting one or more volatilecompounds of interest from the tobacco material with a solvent, whereinthe solvent is a mixture of pentane and diethyl ether; whereindistillation step (ii) and extraction step (iii) are carried outsimultaneously and at a pH of no greater than 2, and wherein the periodduring which both the distillation step (ii) and the extraction step(iii) are carried out is from about 8 to about 20 hours.
 2. The methodaccording to claim 1, wherein the distillation step (ii) and extractionstep (iii) are carried out at a pH of no greater than 2 for the totalperiod of time for which said steps are carried out.
 3. The methodaccording to claim 1, wherein the solvent comprises a mixture of pentaneand ethyl ether in a weight ratio of pentane to ethyl ether of 3:1 to1:1.
 4. The method according to claim 1, wherein the distillation step(ii) and extraction step (iii) are carried out at a pH of from 0.5 to 2.5. The method according to claim 1, wherein the period during which boththe distillation step (ii) and the extraction step (iii) are carried outis from about 8 to about 10 hours.
 6. The method according to claim 1,wherein the distillation step (ii) and the extraction step (iii) arecarried out at a temperature of up to about 130° C.
 7. The methodaccording to claim 1, wherein the tobacco material does not undergo anyheat treatment before distillation step (ii) and extraction step (iii)are carried out.
 8. The method according to claim 1, wherein the one ormore volatile compounds of interest are selected from the groupconsisting of: sugars, sugar esters, amino acids, beta-carotene,violaxanthin, lutein, neoxanthin, phytol, labdanoids, cembranoids,polyphenols, lignin and mixtures thereof.
 9. The method according toclaim 1, wherein the method further comprises a purifying step whichcomprises replacing the solvent used for extraction with another solventsuitable for use in a tobacco product.
 10. The method according to claim9, wherein the solvent suitable for use in a tobacco product is ethanol.